Despite the improvements over the past few years with regard to organ and tissue transplantation, advances in this field continue to be limited by severe shortages in usable human donor organs and tissues. In the past few years, many thousands of transplantation procedures have been performed using allograft tissues, i.e., tissues from a donor animal of the same species as the recipient. However, for any given year, the number of patients which could not obtain such an operation because of the lack of suitable donor tissue greatly exceeds the number of patients which receive the transplants. For example, in 1993, while 18,164 patients received donor organs, a total of 33,394 patients were still on the waiting list for donor organs by the end of that year. In many cases, such as for patients with heart disease, it is unfortunately the case that it is more likely the patient will die while waiting for a suitable donor organ to become available than as a result of complications associated with the transplantation procedure.
In addition, because of the unpredictable availability of organs, transplant surgeries often cannot be scheduled far in advance as would be a routine operation. All too frequently, surgical teams and hospital administrators have to react as soon as a suitable donor organ is located, thereby causing administrative and other difficulties. In the case of heart, liver and lung transplants, for example, if rejection is encountered it will not usually be possible to retransplant unless another suitable donor organ is located within a short period of time, and this is extremely unlikely to occur.
As a result of the shortage in suitable donor tissue, the scientific and medical community has for many years considered the feasibility of xenotransplantation, which is the transplantation of tissue from one species into an individual of a different species. Although there have been numerous attempts to successfully transplant organs or tissues into humans from certain closely related species such as chimpanzees and baboons, those operations have not been without problems, including varying levels of rejection and the requirement of extensive immunosuppressive treatments which can be both rigorous and costly, and which is often accompanied by unwanted side-effects. Moreover, the supply of potentially suitable donor primates is also restricted by other factors including the relatively small numbers of available primate populations, the fact that many potentially acceptable species are on the endangered species list, and other ethical considerations.
Accordingly, the search for suitable animal donors has focused on animals which are not endangered but are in relative ample supply, such as pigs. Unfortunately, xenotransplantation between humans and the most likely donor animal, the pig, has been severely limited due to the severe antibody-mediated rejection that occurs rapidly in humans following transplantation of the xenogeneic organs or tissues. This antibody-mediated rejection, which in its rapid initial stages is known as hyperacute rejection (or HAR), is thought to occur, at least with regard to transplants between pigs and primates, through antibody-mediated complement activation, such as described, e.g, in Dalmasso et al., Amer. J. Pathol. 140:1157-1166 (1992). It is also possible that the alternative pathway of complement is activated in this manner for other species combinations, although at least one paper has suggested that complement regulatory proteins may be species-specific. See Miyagawa et al., Transplantation 46:825-830 (1988).
With regard to hyperacute rejection in transplants of organs from pigs to humans or other primates, the initial stages of this antibody-mediated rejection are usually characterized by thrombosis, hemorrhage and edema, and almost invariably result in a decline in graft function and irreversible rejection within a period of a few minutes to a few hours following transplantation. It has been generally observed that this process is initiated by the binding of xenoreactive natural antibodies (or XNAs) to the carbohydrate structures present on the endothelial cells of the graft which in turn leads to the activation of the complement cascade. See, e.g., Platt et al., Transplantation 50:817-822 (1990).
The prevailing scientific view is that antibody-mediated rejections such as HAR ultimately result in additional rejections instituted by the host's complement defense system after initiation by the XNAs. Complement and its activation are now well known, see Roitt, Essential Immunology (Fifth Edition, 1994), Blackwell Scientific Publications, Oxford, and the activation ascribed to complement (C') depends on the operation of nine protein components (C1 to C9) acting in concert, of which the first consists of three major sub-fractions termed C1q, C1r and C1s. Complement can be activated by the classical or alternative pathway, both of which will now be briefly described.
In the classical pathway, C1 binds to antibody. The C1s subunit acquires esterase activity and brings about the activation and transfer to sites on the membrane or immune complex of first C4 and then C2. This complex has "C3-convertase" activity and splits C3 in solution to produce a small peptide fragment C3a and a residual molecule C3b, which have quite distinct functions. C3a has anaphylatoxin activity which contributes to complement-mediated damage, but otherwise plays no further part in the complement amplification cascade. C3b is membrane bound and can cause immune adherence of the antigen-antibody-C3b complex, so facilitating subsequent phagocytosis.
In the alternative pathway, the C3 convertase activity is performed by C3bB, whose activation can be triggered by extrinsic agents, acting independently of antibody. The convertase is formed by the action of Factor D on a complex of C3b and Factor B. This forms a positive feedback loop, in which the product of C3 breakdown (C3b) helps form more of the cleavage enzyme.
In both the classical and alternative pathways, the C3b level is maintained by the action of a C3b inactivator (Factor I). C3b readily combines with Factor H to form a complex which is broken down by Factor I and loses its hemolytic and immune adherence properties. The classical and alternative pathways are common after the C3 stage. C5 is split to give C5a and C5b fragments. C5a has anaphylatoxin activity and gives rise to chemotaxis of polymorphs. C5b binds as a complex with C6 and C7 to form a thermostable site on the membrane which recruits the final components C8 and C9 to generate the membrane attack complex (MAC). This is an annular structure inserted into the membrane and projecting from it, which forms a transmembrane channel fully permeable to electrolytes and water. Due to the high internal colloid osmotic pressure, there is a net influx of sodium ions and water, leading to cell lysis.
Complement inhibition or restriction factors have been identified which interfere with the action of the complement cascade in such a way as to reduce or prevent its lytic activity; they are often species-specific in that they are relatively ineffectual against complement derived from other species. These factors may be cell membrane bound, or free in serum. Most often they intervene in one of the steps common to both complement activation pathways, however, some factors may be specific to either the classical or the alternative pathway.
In a pig to primate transplant, the host's complement (C') is activated primarily through the classical complement pathway. Preformed xenoreactive natural antibodies (XNAs) circulating in the host's bloodstream recognize and bind to the donor organ, particularly on the luminal surface of the vascular endothelium. Binding of the XNAs serves to trigger the host's complement system. This attack leads to endothelial cell activation, adhesion of platelets and leukocytes, thrombosis and eventual necrosis of the xenograft organ within minutes to a few hours after transplantation. The capillary beds of the transplanted organ appear to be the most sensitive site for attack by the host's complement activity.
It has long been known that complement activation plays a critical role in antibody-mediated rejections such as HAR, as evidenced, e.g., in Gerwurz et al., Transplantation 5:1296 (1967). More recently, the involvement of complement in the antibody-mediated rejection process has been dramatically demonstrated by exogenous inhibition of host complement activity prior to xenotransplantation. For example, several groups have developed experimental methods to inhibit complement by depleting the level of xenogeneic natural antibodies in the host. The xenogeneic natural antibodies are removed either by perfusing the host's blood through a donor organ such as a pig kidney, or by passing the blood over an immunoaffinity column which removes immunoglobulin molecules. See, e.g., Moberg et al., Trans. Proc. 3:538-541 (1971); Fischel et al., Trans. Proc. 24:574-575 (1992); Ye et al., Trans. Proc. 24:563-565 (1992); Agashi et al., Trans. Proc. 24:557-558 (1992). Unfortunately, these methods are not readily transferable to routine use in a clinical transplantation setting.
Alternately, the administration of large amounts of cobra venom factor (see Gerwurz et al. 1967, cited above) or soluble complement receptor (see, e.g., Pruitt et al., Trans. Proc. 24:477-478, 1992) have been shown to be effective in reducing complement activity. Using these methods, at least two independent groups have shown that inhibition of host complement prior to transplantation leads to prolonged xenograft survival. See, e.g., Platt et al., Immunol. Today 11:450-456 (1990); Lexer et al., Trans. Proc. 19:1153-1154 (1987). Xenografts which would normally be rejected in a few hours have been maintained for days and weeks if the host complement is continuously suppressed. In addition, in Miyagawa et al., Transplantation 46:825-830 (1988), the mechanism of discordant xenograft rejection using a guinea pig-to-rat heart graft model was observed to occur by primary activation of complement via the alternative pathway, although the authors suggested that complement regulatory proteins may be species-specific. Complement regulatory proteins, or homologous complement restriction factors, have previously been described, for example, in PCT Application WO 91/05855 issued to Imutran Limited.
Other investigations in this area were conducted by Platt et al. (1990, cited above), who speculated that it might be possible to produce a transgenic pig which directly expressed human decay accelerating factor (DAF), and perhaps other complement regulatory proteins, in the membranes of the pig's endothelial cells. They thought that if such an animal was used as a donor animal, the human complement inhibitor would serve to protect the transplant from human complement. Platt et al. did not describe any specific genetic constructs for accomplishing this goal.
Dalmasso et al. (Transplantation 52:530-533, 1991) suggested engineering a transgenic donor animal, such as the pig, with human membrane-associated C-inhibitor genes to achieve a high level of expression of the corresponding proteins in the endothelial cells of the xenograft.
Consistent with this strategy, White et al., WO 91/05855, prepared transgenic mice bearing a transgene encoding human membrane cofactor protein (MCP) (also known as CD46) or human decay accelerating factor (DAF). The transgene was composed of a Friend spleen focus forming virus 5' long terminal repeat linked to a cDNA encoding the complement inhibitor. However, they did not determine whether these genes were expressed, and, if so, in which tissues, or whether a graft from the transgenic animal would elicit HAR in a discordant animal.
Similarly, Yannoutsos et al., First Int'l Conqr. Xenotr., Abstracts, page 7 (1991), described the development of transgenic mice expressing human DAF and MCP. In this study a series of broad spectrum promoters were used so that it is possible that some of the total complement-inhibitor expression would take place in endothelial cells. Most of their animals appeared to have very low levels of complement-inhibitor expression. Furthermore, they did not confirm that expression in endothelial cells had been achieved, nor did they demonstrate biological function of the transgenically expressed CRP.
Transgenic mice and pigs which contain a human DAF gene have been produced using a partial genomic DNA fragment. (Cary et al., Trans. Proc. 25:400-401, 1993: Cozzi et al., Trans. Proc. 27:319-320, 1995). These animals allegedly exhibited widespread expression of DAF although there appeared to be little expression in hematopoietic tissues. The most consistent expression was observed in vascular smooth muscle, with variable expression in endothelium. It should be noted that the transgenic organs were not tested under transplantation conditions, rather PBMC cells were analyzed using both ELISA and RIA. Therefore no data is presented to suggest that these transgenic organs could prevent HAR in a transplant situation.
Oldham et al., in 1993, presented a talk at the 2nd International Congress on Xenotransplantation in which the production of a human CD59 minigene was announced as well as a modification of this minigene to allow for two cDNA sequences to be inserted into the minigene. Expression of the minigene, containing only the cDNA for CD59, in transgenic mice demonstrated a cell type distribution of CD59 protein expression similar to that seen in human tissues. However, there were problems with regard to this minigene that severely limited its usefulness.
Foder et al., Proc. Nat. Acad. Sci. USA, 91: 11153-57 (1994) sought to produce transgenic mice and swine producing the complement inhibitor CD59 by expressing CD59 under the control of the promoter of the mouse Major Histocompatibility Complex (MHC) Class I gene H2K.sup.b. The latter gene encodes an antigen which is a predominant endothelial cell surface antigen. A CD59 cDNA was cloned into exon I of a 9.0 kb EcoRI genomic restriction fragment of the MHC gene. This fragment included a large 5' sequence, all 8 exons (and the intervening sequences), and a smaller 3' sequence. The authors demonstrated the presence of CD59 in the mouse heart, and in the tails of both mouse and pig; its expression elsewhere was not discussed. Expression was observed on both endothelial and non-endothelial cells.
Eighteen piglets were analyzed by DNA slot blot analysis and one animal was found to have 10-20 copies of the gene while two others contained only one copy of the gene and exhibited no expression or very low and inconsistent levels of expression in peripheral blood mononuclear cells (PBMCs). Low levels of CD59 were seen on a variety of tissues and cell types including fibroblasts, epithelial cells, vascular endothelial cells and smooth muscle cells. Expression of CD59 increased when stimulated with cytokines (which are known to induce the MHC class I promoter). Additionally, since the transgenic organs were not tested in a transplant situation, it is not known whether the organs would be susceptible to complement mediated rejection. Besides the promoters noted above, several other promoters have been used to achieve expression of a transgene in the endothelial cells of a transgenic animal. However, in these prior examples, the transgene was not a complement inhibitor.
Aird et al., Proc. Nat. Acad. Sci. USA, 92: 4567-71 (1995), has generated transgenic mice bearing a chimeric construct that included 487 bp of 5' flanking sequence and the first exon of the Human von Willebrand factor gene fused in-frame to the E. coli lacZ gene. Histochemical analysis of adult tissue demonstrated that LacZ expression was present in the endothelial cells of the blood vessels of the brain, yet activity was absent in the vascular beds of the spleen, lung, liver, kidney, testes, heart and aorta. Certain of the latter beds contain high levels of endogenous von Willebrand factor. This suggested that sequences other than those cloned are necessary for completely authentic expression of von Willebrand factor. One line did not exhibit LacZ activity in brain tissue, and one explanation for this finding is that genomic sequences at or near the site of transgene integration can influence the pattern of expression.
Aird's VWF promoter is of limited value for directing expression to endothelial cells due to its expression being limited to brain tissue. For comparison purposes, Aird et al. fused the E. coli lacZ gene to the promoter of an endogenous thrombomodulin gene by homologous recombination. The resulting transgenic mice exhibited LacZ activity in endothelial cells of all organs, including brain, spleen, lung, liver, heart, kidney and aorta.
Harats et al., J. Clin. Invest., 95:1335 (1995), targeted gene expression to the vascular wall in transgenic mice using the murine preproendothelin-l promoter. Their construct included 5.9 kb of the 5' flanking region, the first exon (with a luciferase reporter gene cloned into the Bg1II site in the noncoding region), and 0.9 kb of the first intron. In all mice, the highest level of expression was in the aorta, and high levels were also noted in other large arteries, in small muscular arteries, and to a lesser extent in capillaries. The level of expression in the veins was lower. Vascular expression was higher in the heart, kidney and lung than in the liver and spleen. Even in the same organ there was substantial variation in vascular expression. Some nonvascular expression was also observed.
Dumont et al., Genes and Development, 8:1897-1909 (1994), created transgenic mice that expressed the lacZ reporter gene under the control of the endothelial receptor tyrosine kinase promoter (tek). This promoter is regulated in a manner which has limited use, i.e., the promoter is turned on during embryonic development and subsequently turned off in the adult (Dumont et al., 1994).
The murine H2K.sup.b class 1 promoter allows for expression in endothelial cells unless stimulated by cytokines, which can result in a higher level of expression (Foder et al., 1994, cited above).
Besides the promoters noted above, there are many others which are associated with genes that encode proteins expressed on the surface of endothelial cells. There is no consensus in the art as to which promoters are most suitable to achieve endothelial expression of a gene of interest, especially in transgenic animals.
Yet another approach to the delivery of C-inhibitors to a transplantable organ was pioneered by Byrne et al., in PCT/US93/08889. Complement inhibitors were specifically expressed in the red blood cells of transgenic animals, which then transfer the proteins to the vascular endothelium of their organs and tissues. Once the organ of interest has been "painted" with complement inhibitor active in the intended recipient, the organ may be safely transplanted.
While this approach appears promising, it takes time to achieve full coverage of the vascular endothelium, so there is a delay before the organ can be used for transplantation. Additionally, once the donor tissue is harvested from the transgenic animal, replacement of the proteins on the surface of the donor tissue will not take place since the organ itself does not produce the protein. The organ would have to be routinely reperfused with the transgenic animal's blood in order to maintain high expression. Therefore, though this approach is promising it has certain limitations. For example, this approach could be beneficial in a situation where expression of the protein in the endothelial tissue is not possible or if it is used to supplement expression levels, which might prove to be beneficial in the first few weeks post-transplantation.
Thorley et al., Transplant. Proc., 27:2177-78 (1995), reported the construction of a CD46 (membrane cofactor protein, MCP) mini-gene. The CD46 gene is more than 45 kb long, precluding the use of a full-length insert in a plasmid vector. The mini-gene was derived from genomic and cDNA sources. It was composed of 7.5 kb of genomic sequence, from 450 5' of exon 1 through to intron 3, and the cDNA sequence corresponding to exons 3-14. The mini-gene was used in the preparation of transgenic mice, however, the pattern of expression of the gene in those mice was not determined.
Accordingly, no satisfactory method has been developed wherein a transgenic animal is produced which expresses a gene for a complement inhibiting protein at a level suitable so that the organs and tissues of the transgenic animals can be useful in xenotransplantation with reduced or eliminated hyperacute rejection. Moreover, no one DNA construct has been manufactured which contains multiple complement inhibitor genes in the same locus that can be used to counteract the problems of hyperacute rejection that occurs following xenotransplantation from distantly related species such as from pig to man.
In addition, as indicated above, other recent studies have now indicated that antibody-mediated rejection, including hyperacute rejection, is initiated by the binding of xenoreactive natural antibodies (or XNAs) to the carbohydrate structures present on the endothelial cells of the graft which leads to the activation of the complement cascade. See Platt et al., Transplantation 50:817-822 (1990). It has thus been shown that the predominant carbohydrate epitope on the xenograft which is recognized by XNAs and thus primarily responsible for antibody-mediated rejection is galactose (.alpha.1,3) galactose, also called the gal epitope or Gal.alpha.(1,3)Gal. See, e.g., Sandrin et al., P.N.A.S. 5 90:11391-11395 (1993) and Transplantation Reviews, 8:134-149 (1994). This epitope is absent in primates, Old World monkeys and humans. See Good et al, Transplant Proc. 24:559-562 (1992) and Galili et al., P.N.A.S. 84:1369-1373 (1987). It is believed that the gal epitope is synthesized in the trans-Golgi by the enzyme Gal.beta.1,4GlcNAc3-.alpha.-D-galactosyltransferase (or ".alpha.(1,3)GT"; EC 2.4.1.51) which catalyzes the addition of galactose to a N-acetyllactosamine (N-lac) core. See Blanken et al., J. Biol. Chem. 260:12927-12934 (1985). Humans, like apes and other Old World monkeys, do not have the gal epitope due to a lack of a functional .alpha.(1,3)GT gene, as shown for example in Galili et al., J. Biol. Chem. 263:17755-17762 (1988) and Larsen et al., J. Biol. Chem. 265:7055-7061 (1990).
Given the specificity of XNAs and previous studies demonstrating that certain aspects of antibody-mediated rejection such as HAR could be delayed by depletion of these antibodies, researchers in this field have attempted to develop ways of achieving non-human donor tissue which lacks the gal epitope, or which has significantly reduced levels of this epitope, such that this tissue will not lead to HAR when transplanted into a human patient. However, the development of an adequate and effective method of achieving reduction of the gal epitope, to allow the production of tissue suitable for xenotransplantation, has proven to be a difficult task because of the complexities surrounding the enzymatic pathways leading to the production of the carbohydrate epitopes in xenogeneic tissues.
For example, the Gal.beta. 2-.alpha.-L-fucosyltransferase (also known as .alpha.(1,2)fucosyltransferase or ".alpha.(1,2)FT"; EC 2.4.1.69) enzyme is involved in the formation of the Fuc.alpha.1,2Gal.beta.- epitope (also known as the H antigen), the characteristic structure of the blood group "O" which is universally accepted in human patients and which is the precursor molecule in the human ABO blood group system. One of the substrate molecules for .alpha.(1,2)FT is N-lac, which is also utilized by .alpha.(1,3)GT as an acceptor of galactose, see Lowe, Semin. Cell Biol. 2:289-307 (1991) and Paulson et al., J. Biol. Chem. 264:17615-17618 (1989). In vitro substrate specificity studies have shown that fucosylated (or sialated) N-lac is a poor substrate for .alpha.(1,3)GT. See Blanken et al., J. Biol. Chem. 260:12927-12934 (1985). However, although there are several fucosyltransferases that are known, the differences in the enzymatic pathways for each enzyme makes it somewhat unpredictable as to how any individual enzyme will perform when expressed in xenogeneic tissues.
Another glycosyltransferase which also uses N-lac as an acceptor is .beta.-galactoside .alpha.(2,6)sialyltransferase (also known as .alpha.(2,6)sialyltransferase or ".alpha.(2,6)ST"; EC 2.4.99.1) which transfers sialic acid residues to N-linked carbohydrate groups of glycoproteins, See, e.g., Lowe, Semin. Cell Biol. 2:289-307 (1991). The enzyme .alpha.(2,6)ST is expressed in human endothelial cells, B cells, etc., and therefore should not be antigenic. See Dorken et al., in Leukocyte Typing IV, White Cell Differentiation Antigens, Knapp et al., eds., Oxford University Press, pp. 109-110 (1989) and Hanasaki et al., J. Biol. Chem. 269:10637-10643 (1994). In fact, due to its negative charge, sialic acid has sometimes been shown to inhibit activity of the alternative pathway of human complement. See Fearon, P.N.A.S. 75:1971-1975 (1978). However, once again, there is great variance among sialyltransferases, and the effectiveness of any particular sialyltransferase may vary from one to another.
Still another of the glycosyltransferases that may be involved in pathways that may compete with the .alpha.(1,3)GT enzymatic pathways is .beta.1,3 N-acetylglucosaminyltransferase (or ".beta.(1,3)NAGT"), which is described in Kornfield et al., Ann. Rev. Biochem. 54:631-664 (1985). However, the complex enzymatic pathways associated with .beta.(1,3)NAGT are far from clear, and it has not been disclosed or suggested that such an enzyme would be useful in a method of reducing the level of the gal epitope in xenogeneic donor tissue, organs, cells or non-viable components that would be useful in xenotransplantation.
Moreover, it has still not been previously shown that the expression of any other glycosyltransferase, such as human .alpha.(1,2)FT or .alpha.(2,6)ST, in transgenic animals such as pigs and mice can produce suitable antigens on endothelial cells of multiple organs so as to achieve a reduction in XNA binding (and subsequent complement activation) and allow large scale production of usable donor tissue and organs from transgenic animals which will have reduced or eliminated antibody-mediated rejection when transplanted into human patients. Additionally, previous researchers have deemed it unlikely that techniques involving use of sugar structures such as sialyltransferase or fucosyltransferase to produce transgenic animals with altered expression of the gal epitope which would be useful in xenotransplantation. See Cooper et al., Immunological Reviews 141:31-57 (1994). Again, due to the complexity of the sialyltransferases and fucosyltransferases and the substantial differences between individual members of these groups (see, e.g., Kitagawa et al., J. Biol. Chem., 269:17872-17878, 1994), the potential usefulness of any particular members of these groups has remained uncertain and unpredictable, and indeed many of these enzymes may be undesirable for xenotransplantation.
In addition, it has not been shown that a transgenic animal can be prepared which expresses multiple types of genes which can effectively mask or reduce the level of the antigenic gal epitope while at the same time reduce the possibility or likelihood of activating the complement cascade following xenotransplantation. Similarly, it has also not been previously shown that a gene for a complement inhibitor can be used in combination with a gene that masks or reduces the level of the gal epitope so as to produce transgenic animals suitable for use in transplantation. Finally, it has not previously been accomplished to place multiple genes on a single DNA construct which can be transfected into a donor animal and expressed so as to provide an increased level of protection against antibody-mediated rejection when organs, tissues, cells or non-viable components from the donor animal are transplanted into a human patient.